Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of angiogenic proliferative skin[unreadable] lesions (KS), as well as of several rare B-cell lymphomas (PEL and MCD). KS is particularly prevalent in[unreadable] AIDS patients and has now become the most common of all tumors in Southern Africa. In previous studies,[unreadable] we have: (A) Mapped and characterized patterns of KSHV mRNA and protein expression in latent and TPA-induced[unreadable] lytic PEL cell lines by primer extension, northern blots, sequencing of isolated cDNA clones, and[unreadable] indirect I FA with specific antibodies; (B) Established a biological assay system for primary de novo infection[unreadable] by KSHV in contact-inhibited adult dermal microvascular endothelial cells (DMVEC) in culture, which[unreadable] produces a proliferative spindle cell conversion phenotype associated with stable maintenance of a LANA1-[unreadable] positive episomal latent state, together with low level lytic cycle reactivation, features that closely mimic[unreadable] those found in nodular KS lesions; (C) Carried out gene array and real-time RT-PCR analyses of KSHV-infected[unreadable] compared to uninfected DMVEC, to identify and confirm a set of 27 highly up or down-regulated[unreadable] endothelial cell (EC) mRNAs; (D) Detected the absence or shutoff of several key vascular EC proteins in[unreadable] KSHV-infected spindle cells by IFA, including PECAM1/CD31, VWF, VE-Cadherin, and VCAM1; and (D)[unreadable] Demonstrated by immunohistochemistry (IHC) that essentially all LAN A1-positive fascicular and perivascular[unreadable] spindle cells of nodular KS also lack CD31 and VWF, but are consistently PROX1-positive.[unreadable] Our new goals in this proposal are to further evaluate the altered protein expression patterns and EC[unreadable] functions of both KS and cultured KSHV-infected spindle cells; to define novel features of the viral[unreadable] transcription profiles in KSHV-infected spindle DMVECs, and to assess the relative contributions of three[unreadable] KSHV proteins vFLIP, vMIR1 and vMIR2 to the apparent conversion of functionally intact metabolic vascular[unreadable] EC into proliferating pre-angiogenic EC with lymphatic features. The three Specific Aims include: (I) Further[unreadable] IHC analysis of the patterns of expression of key altered EC proteins in KSHV-infected spindle and[unreadable] neovascular vessel wall EC in KS tissue, and evaluating the functional status of KSHV-infected spindle[unreadable] DMVEC, including potential loss of normal metabolic EC functions such as forming 3-D tubules or the ability[unreadable] to respond to inflammatory cytokines; (II) Mapping novel mRNA transcripts, splicing patterns and promoters[unreadable] of KSHV that are associated with maintenance of latency or early lytic cycle reactivation in DMVECs,[unreadable] including generation and analysis of cDNA libraries and use of a new generation KSHV viral and cellular[unreadable] gene chip array; (III) Using high efficiency ectopic expression of isolated viral genes, siRNA interference and[unreadable] BAC knockout insertion or deletion genetic approaches, to identify processes that may specifically target key[unreadable] DMVEC proteins for down-regulation by either the KSHV vFLIP anti-apoptotic and NFkB-activating latency[unreadable] protein, or at the post-translational level by the KSHV vMIR and vMIR2 membrane ubiquitin E3 ligases.